Sulphamidase activity in leucocytes, cultured skin fibroblasts and amniotic cells: diagnosis of the Sanfilippo A syndrome with the use of radiolabelled disaccharide substrate.
1. Sulphamidase activity was assayed by incubation of the radiolabelled disaccharide O-(alpha-2-sulphamino-2-deoxy-D-glucopyranosyl)- (1 leads to 3)-L-[6-3H]idonic acid with homogenates of leucocytes and cultured skin fibroblasts and concentrates of urine derived from normal individuals, patients affected with sulphamidase deficiency disorder [mucopolysaccharidosis type IIIA (MPS IIIA): the Sanfilippo A syndrome], parents of such patients and patients affected with other mucopolysaccharidoses and lysosomal enzyme deficiencies. 2. The assay clearly distinguished affected homozygotes from normal controls, heterozygotes and other mucopolysaccharidoses types. 3. Sulphamidase displayed remarkable thermal stability; reaction rates were constant for at least 24 h at 60 degrees C for leucocyte and 20 h at 37 degrees C for cultured fibroblast preparations. Apparent Km values for fibroblast sulphamidase were 71 mumol/l at 37 degrees C and 100 mumol/l at 50 degrees C; the corresponding Vmax, values were 21 and 72 pmol min-1 mg-1 of protein respectively. An incubation temperature of 60 degrees C was used for the routine assay of sulphamidase activity in leucocytes, urine and amniotic supernatant preparations. The specific activities of fibroblast and amniotic cell sulphamidase, assessed at incubation temperatures of 37 degrees C, were more than 10-fold the leucocyte enzyme activity at 60 degrees C. 4. We recommend the use of radiolabelled disaccharide substrate for the assay of sulphamidase in leucocytes, skin fibroblasts and urine, for the routine enzymic detection of the sulphamidase deficiency disorder of the Sanfilippo A syndrome.
D000650:Amnion / Q000201:enzymology; D002478:Cells, Cultured; D004796:Clinical Enzyme Tests; D004187:Disaccharides; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D006867:Hydrolases / Q000378:metabolism*; D007962:Leukocytes / Q000201:enzymology; D009083:Mucopolysaccharidoses / Q000175:diagnosis* / Q000201:enzymology; D009084:Mucopolysaccharidosis III / Q000175:diagnosis* / Q000201:enzymology / Q000652:urine; D012867:Skin / Q000201:enzymology
D004187:Disaccharides; C032935:O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-(1-3)-L-idonic acid; D006867:Hydrolases; C022708:N-sulfoglucosamine sulfohydrolase